Electrophoresis, Acrylamide Gel

Surfactants are amphophilic molecules, which consist of a hydrophobic carbohydrate part and a hydrophilic head group. In capillary zone electrophoresis (CZE), different types, i.e., anionic, cationic, but also neutral, tensides are employed. The ability of such molecules to interact with ionic and nonionic species has been used in ion chromatography and, in particular, in SDS-poly-(acrylamide) gel electrophoresis (PAGE) (15). 

Thin-layer chromatography (75) and sodium dodecylsulfate-(poly)acrylamide gel electrophoresis (SDS-PAGE) are helpful for analyses of the lipid and protein composition, respectively. Size-exclusion chromatography allows estimation of the size distribution of the (proteo)liposomes and crude fractionation of the material as reviewed in Ref. 76. Accurate determinations of size distributions require analyses by static or dynamic... 

Only a few bacterial and viral sialidases have been purified to high purity or even to protein homogeneity."0 Complete purification of sia-lidase on a preparative scale from the culture filtrate ofC. perfringens was achieved111 by using poly(acrylamide) gel-electrophoresis as the final purification step (see Section VI,1). It is necessary that such purified sialidases be available, as the presence of proteases or other gly-cosidases in the enzyme preparations would lead to severe errors, not only in studies of substrate specificity, but also in cell biological and medical studies (see Sections VI and VII). 

Sialyltransferases can be solubilized from their subcellular site by using detergents, and be purified by affinity chromatography on, for example, CDP-6-aminohexanol-agarose,267 as already described. Solubilization of frog- and rat-liver sialyltransferases by means of Triton X-100 has been described.27 Soluble sialyltransferase occurs in colostrum, and is also present in small quantities in normal blood-serum. From the latter source, the enzyme was purified 300-fold by poly (acrylamide) gel-electrophoresis.2 ... 

Once a DNA molecule has been cleaved into fragments, a particular fragment of known size can be enriched by agarose or acrylamide gel electrophoresis or by HPLC (pp. 92, 90). For a typical mammalian genome, however, cleavage by a restriction endonuclease usually yields too many different DNA fragments to permit isolation of a particular fragment by electrophoresis or... 

Nagasawa, T., Kiyosawa, I., Kuwahara, K. and Ganguly, N. C. 1973. Fractionation of buffalo milk casein by acrylamide gel electrophoresis and DEAE cellulose column chromatography. J. Dairy Sci. 56, 61-65. 

The biosynthesis in yeast of two enzymes that are D-mannoproteins has been studied. A membrane-associated isozyme of invertase (EC 3.2.1.26) has been shown to be a precursor of the external invertase.190 Its molecular weight, as determined by SDS-poly(acrylamide) gel electrophoresis, is 50,000, that is, smaller than that of the external invertase, and it correlates well with the presence of only the inner-core sugars of the final form. It is strictly bound to membranes, possibly those of the endoplasmic reticulum, and it can be completely split191 by endo-/3-N-acetylglucosaminidase H (EC 3.2.1.30). The addition of tunicamycin, which specifically inhibits formation of d-GIcNAc-PP-DoI, inhibits synthesis of external invertase, as well as further formation of the membrane-associated form, which completely disappears after addition of the antibiotic.190 In these aspects, the synthesis of this extracellular enzyme follows the pathway for secreted glycoproteins in animal systems. 

The temperature dependence of the friction coefficient of poly(acrylamide) gel was studied at two different concentrations of acrylamide (AAm) and N,N -methylenebisacrylamide (Bis) AAm Bis = 1.24 M 22.4 mM and AAm Bis = 693 mM 7 mM. The weight concentrations of these sample gels was about 8.8 and 5 wt.%, respectively. The composition of the latter gel is almost the same as the composition typically recommended for acrylamide gel electrophoresis. The friction coefficient of these gels was measured at a fixed pressure of 5.88 x 103 Pa, which corresponds to 60 cm of the height of the water column. The temperature was varied from about 0 to 60 °C. 

Fig. 2.4. Vector characterization. (A). SDS acrylamide gel electrophoresis of a rAAV-UF-11 vector. Approximately 2.5 x 1011 drp were loaded on a 10% SDS-polyacrylamide gel. The three capsid protein bands in the correct stoichiometry are visualized by silver staining. (B) Infectious center assay of a rAAV-CB-hAAT stock when probed for the transgene each spot represents a vector infected cell. (C) Dot blot analysis of the rAAV stock to determine the titer of DNase-resistant particles of rAAV. Amounts (ng) refer to a serial dilution of the packaged rAAV vector plasmid used to construct the standard curve. The stock has a calculated titer of 4.3 x 1013 drp/ml.

From a strict biochemical point of view a clear-cut definition of the role of the liver in the biosynthesis of any particular plasma protein can be made only when the particular protein has been clearly and cleanly isolated, as in the case of fibrinogen. The practical difficulties of effecting such isolations on a small scale from isotopic labeling studies of the plasma proteins, such as we have described, seriously militate against such a detailed demonstration at present. The use of fractionation techniques with greater resolving power such as acrylamide gel electrophoresis already show some promise in our laboratory toward affording a more definitive picture of the biosynthetic role of the liver and the nonhepatic tissues in plasma protein production. 

To still further determine the composition of the channels, the PHB-Ca2+-PolyP complexes were reconstituted. PHB was recovered from E. coli and carefully purified, and Ca2+-PolyP was prepared from commercial sodium PolyP and calcium chloride. Single-channel currents similar to those described above were obtained by three different experimental procedures, as described by Reusch et al. (1995). The chain length of chemically synthesized PolyP was determined by acrylamide gel electrophoresis to be in the same range (55-65 residues) as in the E. coli complexes (Castuma et al., 1995). 

Schagger, H. and von Jagow, G. (1987) Tricinesodium dodecyl sulfatepoly-acrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDA. Anal. Biochem. 166, 368-379. 

On treatment with 1,4-dithiothreitol or 2-mercaptoethanol in dodecyl sodium sulfate, components II and III both yielded199,586 subunits of molecular weight 31,000. Poly(acrylamide) gel-electrophoresis, in the presence of 1% of dodecyl sodium sulfate alone, gave586 a component in the range of 60,000. Amino acid analysis (see later) showed two half-cystine residues per subunit. Direct titration of components II and III with 5,5 -dithiobis(2-nitrobenzoic acid), in the absence or presence of 8 M urea or dodecyl sodium sulfate, gave199,588... 

Bausch and Poretz purified M. pomifera agglutinin on insolubilized polyleucyl, hog-gastric mucin.634 The immunochemically homogeneous protein migrated as a single component at acidic or alkaline pH in poly(acrylamide) gel electrophoresis, and focused as a single band... 

A water-soluble, 50%-methanol-soluble, acidic glycoprotein was isolated from rat brain by affinity chromatography on con A-Sepharose.855 The glycoprotein, pure by poly(acrylamide) gel-electrophoresis at pH 8.8, had an apparent molecular weight of 14,500 1,400 by dodecyl sodium sulfate gel-electrophoresis. No analysis for carbohydrate was reported.855... 

Figure 5. SDS-acrylamide gel electrophoresis of brush border membrane proteins from rats of adequate zinc status (0), following a 4-day zinc depletion (4), and following an 8-day zinc depletion (8). Proteins migrating with approximate molecular weights of 63,000 and 45,000 daltons are more apparent after 4 and 8 days of dietary zinc restriction.

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